I perform double digestions for all three plasmids:1)Vector: KpnI HF + XbaI in buffer4 1h at 37C, then add phosphatase and incubate 15min at 37C and then incubate 20min at 65C to inactivate phosphatase2)Insert1: KpnI HF + DraIII in buffer1 1h at 37C3)Insert2: XbaI + DraIII in buffer3 (75%activity of XbaI) 1h at 37C

Then I run digested mixes on agarose gel and cut out the bands of interest (I can see that vectors for Insert1 and Insert2 are nicely digested, but for vector I cannot say since the cut part is very small – it runs almost the same as undigested vector).I purify the bands of interest using Qiagene Kit and then perform ligation reaction using ratio 1:3:3 (vector:insert1:insert2).I also tried ligation without gel purification of bands (so I don’t expose DNA to UV light), in which case I just purify digestion mixes using DNA purification Kit (Qiagene).I always get very small number of colonies, run colony PCR and get inconclusive results (for PCR for insert1 I don’t get anything, for PCR for insert2 I get a band of interest and unspecific bands). I send it for sequencing and get nothing!Does anyone have any advice re three part ligations? Is it much more complicated than 2 part ligation?

You should not need sequencing to tell if you have good ligations. You can simply miniprep and examine the length of the insert. I don't see anything wrong with your approach, but I would use a 1:1:1 ratio (this is unlikely to be the problem). I would guess the main problem would be background from uncut or partially cut vector, and would expect vector only colonies to dominate.

You should not need sequencing to tell if you have good ligations. You can simply miniprep and examine the length of the insert. I don't see anything wrong with your approach, but I would use a 1:1:1 ratio (this is unlikely to be the problem). I would guess the main problem would be background from uncut or partially cut vector, and would expect vector only colonies to dominate.

Right. Then I'll try o/n digestion of the vector and see how it works.Thank you!